PURIFICATION OF zyxwvutsrq PHOSPHATIDYLGLYCEROPHOSPHATE SYNTHETASE Membrane-Associated Phosphatidylglycerophosphate Synthetase from zyx Escherichia coli: Purification by Substrate Affinity Chromatography on Cytidine 5’-Diphospho- 1,2-diacyl-sn-glycerol Sepharose? Takashi Hirabayashi,l Timothy zyxwvutsrq J. Larson, and William Dowhan* ABSTRACT: The membrane-associated cytidine 5’-diphos- pho- 1,2-diacyl-sn-glycerol (CDPdig1yceride):sn-glycerol- 3-phosphate phosphatidyltransferase (EC 2.7.8.5) from Escherichia coli has been solubilized with Triton X-100 and purified 6000-fold to 85% of homogeneity. The major purifi- cation was attained using several modifications of the CDP- diglyceride Sepharose affinity chromatography system de- scribed by Larson et al. (Larson, T. J., Hirabayashi, T., and Dowhan, W. (1976), Biochemistry zyxwvutsr 15, 974). The native en- zyme in Triton X-100 had an apparent molecular weight of over 200 000, as judged by Sepharose 6B gel filtration. The apparent size of the native enzyme appeared to be due to its association with Triton X-100, as judged by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and the lack of affinity for ion-exchange resins. The minimum subunit molecular weight of the enzyme, determined by sodium do- The enzymes responsible for phospholipid biosynthesis are involved in the processes of membrane biogenesis and cell growth. Due to the particulate nature of most of these enzymes, they are also important functional components of many membranes. Currently, little is known concerning the control of this important group of enzymes or their physical, chemical, and enzymological properties. These types of studies have been hampered by the lack of rapid procedures for obtaining sig- nificant amounts of highly purified enzymes for investigation. Within this group of enzymes, only the phosphatidylserine decarboxylase of Escherichia coli has been purified to near homogeneity (Dowhan et al., 1974). This and the following paper in this issue (Larson and Dowhan, 1976) describe the purification and properties of the CDP-diglyceride1:sn-glycerol-3-phosphate phosphatidyl- transferase (EC 2.7.8.5, PGP synthetase) and CDP-diglyc- eride:L-serine 0-phosphatidyltransferase (EC 2.7.8.8, phos- phatidylserine synthetase), respectively, from E. coli. Since these two enzymes form the branch point in phospholipid metabolism which leads to the formation of either a zwitter- ionic phospholipid (phosphatidylethanolamine) or acid phos- pholipids (phosphatidylglycero1 and cardiolipin), they may be + From the Department of Biochemistry and Molecular Biology, Uni- versity of Texas Medical School, Houston, Texas 77025. zyxwvutsrqp Receiaed June 9, 1976. This work was supported by grants from the National Institutes of Health (GM 20478) and the Robert A. Welch Foundation (AU-599). Material for this manuscript will be used for partial fulfillment of the re- quirements for the Ph.D. of T.J.L. Present address: Central Research Institute, Suntory, Ltd., Shima- moto-cho, Osaka, Japan. ’ Abbreviations used are: CDP-diglyceride and dCDP-diglyceride, cytidine and deoxycytidine 5’-diphospho- I .2-diacyl-sn-glyceroI, respec- tively; PGP, 3-sn-phosphatidyl- I’-sn-glycero-3’-phosphate; DEAE, di- ethylaminoethyl. decyl sulfate polyacrylamide gel electrophoresis, was 24 000. This low molecular weight is consistent with the stability of the enzyme to heat, urea, or sodium dodecyl sulfate denaturation. The purified enzyme had an absolute requirement for mag- nesium ion (KM = 50 mM) and Triton X-100 (0.5-6%) for activity when either CDP-diglyceride or dCDP-diglyceride was used as substrate. Kinetic analysis of the enzymatic reaction indicated an ordered sequential Bi-Bi reaction with the lip- onucleotide forming a dead-end complex at high concentration, which inhibited both the forward and reverse reactions. The enzyme would not hydrolyze the pyrophosphate bond of its lipid substrate or the phosphate esters of its lipid product but would catalyze a cytidine 5’-monophosphate dependent ex- change reaction between glycero-3-phosphate and phospha- tidylgl ycerophosphate. involved in controlling the charge density of the membrane surface, as well as the absolute levels of these lipids. The phosphatidylserine synthetase is a cytoplasmic or possibly a ribosomal-associated enzyme (Raetz and Kennedy, 1972 and 1974), which may play an important role in the coordination of phospholipid metabolism with other cellular processes. The PGPsynthetase in both E. coli (Chang and Kennedy, 1967a) and Bacillus licheniformis (Larson et al., 1976) is associated tightly with the membrane. The PGP synthetases can be sol- ubilized from the membrane using nonionic detergents but are resistant to purification by ion-exchange chromatography in the presence of these detergents. Larson et al. (1976) were able to partially purify the PGP synthetase from B. licheniformis by CDP-diglyceride Sepharose affinity chromatography. Using a similar affinity chromatography technique, we have purified the membrane-associated PGP synthetase from E. coli 6000-fold to near homogeneity. We report the minimum mo- lecular weight of the enzyme and discuss the interaction of the native enzyme with detergent. The effect of substrate, deter- gent, and divalent metal ion on enzymatic activity is also re- ported. Materials and Methods Reagents. All chemicals were reagent grade or better. sn- Glycero-3-phosphate was purchased from Calbiochem. DEAE-Sephadex (A-50) and Sepharose 4B and 6B were products of Pharmacia. Radiochemicals were purchased from Amersham/Searle. Triton X-100 was a product of Rohm and Haas. Sodium dodecyl sulfate (99% dodecyl) was purchased from Bio-Rad. Ovalbumin and hemoglobin were supplied by Sigma. Bovine serum albumin and erythrocuprein were pur- chased from Miles Laboratories. Escherichia coli B (3/4 log), grown on rich medium, was purchased as the frozen cell paste from Grain Processing, Muscatine, Iowa. BIOCHEMISTRY, VOL. 15, NO. 24, 1976 5205